To establish a life-long infection, herpesviruses have evolved means of subverting normal host defenses. We, and others, have found that Kaposi's Sarcoma associated herpesvirus (KSHV) encodes two proteins, MIR1 and MIR2, that prevent antigen presentation by MHC-I molecules. These proteins decrease MHC-I expression on the surface of the infected cell, thus preventing recognition by cytotoxic T lymphocytes (CTLs). These MIR proteins act as E3 ubiquin ligases of the ubiquitin pathway. They promote ubiquitination of the MHC-I intracytoplasmic domain, a signal that targets MHC-I molecules for internalization from the cell surface and degradation by the lysosomes. . Lysine residues and the N-terminus were long thought to be the exclusive sites of ubiquitin attachment on substrates. Unexpectedly, we recently observed that the MIR proteins have the unique ability to ubiquitinate their substrates on cysteine residues. Whether this novel form of ubiquitination is specific to a few highly specialized E3s (and thus has evolved as a functional requirement) or if it is a more generalized process is still an open question. In Aim#1, we propose to further study this novel form of ubiquitination and to identify MIR substrates regulated through this mechanism. In Aim#2, we propose to characterize how the MIR proteins regulate the ubiquitination of their substrates. This will not only shed light into the biology of the viral MIR proteins but more broadly into the mechanism of polyubiquitination by cellular E3 ubiquin ligases.